Programmed death-ligand 1 (PD-L1) has been shown to negatively regulate immune responses via its interaction with PD-1 receptor. In this study, the effects of PD-L1 on intestinal inflammation was investigated using two murine models of inflammatory colitis induced by dextran sulfate sodium (DSS) and CD4+ CD45RBhigh T cell transfer. The anti-colitis effect of adenovirus expressing nonlytic Fc-conjugated PD-L1 (Ad/PD-L1-Fc) and recombinant PD-L1-Fc protein was evaluated in DSS-treated wild-type and Rag-1 knockout (KO) mice. It was examined about differentiation of T helper cells, frequency of innate immune cells, and cytokine production by dendritic cells (DCs) in the colon from DSS-treated mice after PD-L1-Fc administration. In Rag-1 KO mice reconstituted with CD4+ CD45RBhigh T cells, treatment effect of PD-L1-Fc protein was assessed during development of colitis. This study demonstrated for the first time that administration of PD-L1-Fc, either in the form of recombinant protein or by utilizing adenoviral vector as a delivery vehicle, could ameliorate DSS-induced colitis. The therapeutic effect of PD-L1-Fc in DSS colitic mice was associated with decreased frequency of interleukin (IL)-17A-producing CD4+ T cells and increased IFN--producing CD4+ T cells in the colon. The anti-colitic effect of PD-L1-Fc treatment was also observed in DSS-treated Rag-1 KO mice, indicating lymphoid cell independency. PD-L1-Fc modulated cytokine secretion by colonic DCs and the effects was dependent on PD-1 expression upon toll-like receptor (TLR) stimulation. Furthermore, PD-L1-Fc protein could significantly reduce the severity of colitis in CD4+ CD45RBhigh T cell-transferred Rag-1 KO mice. Based on the protective effect of PD-L1-Fc against DSS-induced acute and T cell-induced chronic colitis, these results suggest that PD-1-mediated inhibitory signals have a crucial role in limiting the development of colonic inflammation. This implicates that PD-L1-Fc may provide a novel therapeutic approach to treat inflammatory bowel disease.